曲妥珠单抗对SW-620人结肠癌细胞增殖凋亡的影响及其与奥沙利铂协同作用的研究

Effect of trastuzumab on proliferation and apoptosis of SW-620 human colon cancer cell and its synergistic effect with oxaliplatin

目的观察不同浓度曲妥珠单抗单独或联合奥沙利铂对SW-620人结肠癌细胞增殖、凋亡及细胞周期的影响,并探讨其作用机制。方法体外培养SW-620人结肠癌细胞。(1)细胞增殖实验:将细胞分为2个大组:曲妥珠单抗组和曲妥珠单抗+奥沙利铂组,每个大组内设8个浓度小组(每个小组设5个复孔),曲妥珠单抗组的浓度依次为0、0.001、0.01、0.1、1、10、100及1 000μg/mL,对应的曲妥珠单抗+奥沙利铂组曲妥珠单抗的浓度与前相同,不同之处在于均加入了奥沙利铂(10μmol/L)。采用CCK-8法检测各组细胞的吸光度(A)值。(2)细胞凋亡实验:组别设置同增殖实验,只是曲妥珠单抗的浓度仅包括0、0.1、1及10μg/mL。采用流式细胞仪检测各组细胞的细胞凋亡率及细胞周期分布。(3)人表皮生长因子受体2(her-2)蛋白表达检测:分别以0、100及1 000μg/mL曲妥珠单抗,以及0、1及10μg/mL曲妥珠单抗联合奥沙利铂(10μmol/L)处理SW-620细胞,采用Western blot法检测各组细胞中her-2蛋白的表达。结果 (1)细胞增殖实验:同种浓度下曲妥珠单抗+奥沙利铂组的A值均低于曲妥珠单抗组(P<0.05),且随着曲妥珠单抗浓度的增加,A值在逐渐降低。(2)细胞凋亡实验:同种曲妥珠单抗浓度下,曲妥珠单抗组+奥沙利铂组的细胞凋亡率均较曲妥珠单抗组高(P<0.05)。流式细胞检测:不同浓度曲妥珠单抗+奥沙利铂处理后,随浓度增加,G_1期细胞比例呈下降趋势,S期细胞比例呈上升趋势,且呈剂量依赖性;至1μg/mL和10μg/mL时,与曲妥珠单抗组比较,曲妥珠单抗+奥沙利铂组的G_1期细胞比例较低,S期细胞比例较高(P<0.05);在0.1、1和10μg/mL浓度的曲妥珠单抗条件下,曲妥珠单抗联合奥沙利铂组的G_2期细胞比例较曲妥珠单抗组升高(P<0.01)。(3) her-2蛋白的表达水平:1、100及1 000μg/mL曲妥珠单抗组细胞中her-2蛋白的表达水平逐渐降低,3组间两两比较差异均有统计学意义(P<0.05);0、1及10μg/mL曲妥珠单抗+奥沙利铂组中her-2蛋白的表达水平也逐渐降低,3组间两两比较差异也有统计学意义(P<0.05)。结论高浓度曲妥珠单抗可抑制SW-620人结肠癌细胞的增殖,诱导其凋亡。曲妥珠单抗和奥沙利铂具有协同抑制细胞增殖、促进细胞凋亡的作用。

Objective To observe the effects of different concentrations oftrastuzumab alone or in combination with oxaliplatin on proliferation,apoptosis,and cell cycle of SW-620 human colon cancer ceil,and to explore its mechanism.Methods SW-620 human colon cancer cells were cultured in vitro.①Cell proliferation experiment:the ceils were divided into two large groups:trastuzumab group and trastuzumab combined with oxaliplatin group.There were eight concentration groups in each large group (five holes for each group).The concentration of the trastuzumab group was 0,0.001,0.01,0.1,1,10,100,and 1000μg/mL,corresponding to the trastuzumab combined with oxaliplatin group. The concentration of the antibiotic was the same as before,except that oxaliplatin (10μmol/L)was added.The absorbance (A)value of each group was measured by CCK-8 method.②Apoptosis experiment:the same proliferation experiment was performed in the group,except that the concentrations of trastuzumab only included 0,0.1,1 and 10μg/mL.Flow cytometry was used to detect the proportion of apoptotic cells and cell cycle distribution in each group. ③Determination of human epidermalgrowth factor receptor-2(her-2).The SW-620 cells were divided into two large groups,the concentration of trastuzumab group concluded 0,100,and 1000μg/mL,as well as the concentration of trastuzumab in the trastuzumab combined with oxaliplatin group concluded 0,1,and 10μg/mL.Expressions of her-2 protein in SW-620 cells were detected by Western blot method.Results ①Cell proliferation assay:the A values at 100μg/mL and 1000μg/mL were significantly lower than that at 0μg/mL (P<0.05).At the same concentration,the A value of the trastuzumab combined with oxaliplatin group was lower than that of the trastuzumab group (P<0.05),and the A value gradually decreased with the increase of the concentration of trastuzumab.②Apoptosis experiment:the proportion of apoptotic cells in the trastuzumab combined with oxaliplatin group was higher than that in the trastuzumab group (P<0.05)at the same concentration of trastuzumab.Flow cytometry:after treatment with different concentrations of trastuzumab combined with oxaliplatin,ceils in GI phase showed a downward trend,and cells in S phase showed an upward trend in a dose-dependent manner.At 1and 10μg/mL concentration oftrastuzumab,the trastuzumab combined with oxaliplatin group significantly reduced the proportion of cells in the G1 phase of SW-620 cell cycle compared with the trastuzumab group,but S phase ratio was higher (P<0.05).The proportion of G1 phase cells was significantly higher in the trastuzumab combined with oxaliplatin group than the trastuzumab group at 0.1,1and 10μg/mL concentrations of trastuzumab (P<0.01).③Expressions of her-2 protein:the expression level of her-2 protein gradually decreased at 1,100, and 1000μg/mL trastuzumab group (P<0.05).The expression levels of her-2 protein in 0,1 and 10μg/mL trastuzumab combined with oxaliplatin group also gradually decreased (P<0.01).Conclusions High concentration oftrastuzumab can inhibit the proliferation of SW-620 human colon cancer cells and induce apoptosis.Trastuzumab combined with oxaliplatin has synergistic effect on inhibiting cell proliferation and promoting apoptosis.